Myeloma cell and ovarian cancer cell surface glycoproteins, antibodies thereto, and uses thereof

ABSTRACT

The present invention is directed to cell surface antigens found on myeloma cells and on ovarian cancer cells, and monoclonal antibodies and binding fragments thereto capable of being used for therapeutic, diagnostic, and cell purification purposes.

FIELD OF THE INVENTION

The application is related to new surface glycoproteins of human myelomacells and human ovarian tumor cells, monoclonal antibodies thereto, andmethods of diagnosis and treatment of myeloma and ovarian cancer basedthereon.

BACKGROUND OF THE INVENTION

Multiple myeloma (MM) embodies a plasma cell disorder characterized byneoplastic proliferation of a single clone of plasma cells engaged inthe production of a monoclonal immunoglobulin, usually monoclonal IgG orIgA. MM accounts for 1% of all malignant disease and slightly more than10% of all hematologic malignancies. The annual incidence of multiplemyeloma is 4 per 100,000. The annual incidence is linked to agingpopulation. The median age of patients at the time of diagnosis is 61years. MM is most common in men, and in individuals of African ancestry.

MM remains a disease for which a cure is a rarity. Most patients succumbto their disease within 36-48 months from the time of diagnosis. Thelimitations of effective therapy for MM are primarily associated withthe low cell proliferation rate and multi-drug resistance. Therapy formultiple myeloma includes induction, maintenance, and supportiveaspects. The induction portion of the treatment aims at reducing thetumor volume, and achieving a plateau phase. Different drugs, andtreatment modalities such as bone marrow transplantation has beenentertained, and used without a significant impact on the disease or theoverall survival. Supportive care in multiple myeloma has advancedsignificantly over the past few years. Growth factor support witherythropoietin replacement and GM-CSF for stimulating the WBC is a safeand effective method of decreasing or preventing the occurrence or theseverity of neutropenia. Also high dose chemotherapy followed byautologous bone marrow or peripheral blood progenitor cell (PBMC)transplantation has recently increased the complete remission rate andremission duration. However, overall survival has only been slightlyprolonged, and no evidence for a cure has been obtained. All patientsultimately relapse even under maintenance therapy with interferon-α(IFN-α) alone or in combination with steroids. Adoptive immunotherapyrather than active vaccination may prove to be a more effective therapyfor MM patients. There are relatively known few surface antigens on theplasma cells that are suitable for antibody-directed treatment. Possiblemolecules include HM1.24, CD38, ICAM-1 (CD54), CD40, CD45, CD20, andsyndecan 1.

To-date there are no exclusive markers reported for MM. CD20, CD38, CD56and CD130 are all markers that are expressed on normal B, T or NK cells.

Ovarian cancer is the fifth leading cause of cancer deaths among U.S.women and has the highest mortality of any of the gynecologic cancers.It accounted for an estimated 26,600 new cases and 14,500 deaths in1995. The overall 5-year survival rate is at least 75% if the cancer isconfined to the ovaries and decreases to 17% in women diagnosed withdistant metastases. Symptoms usually do not become apparent until thetumor compresses or invades adjacent structures, ascites develops, ormetastases become clinically evident. As a result, two thirds of womenwith ovarian cancer have advanced (Stage III or IV) disease at the timeof diagnosis. Carcinoma of the ovary is most common in women over age60. Other important risk factors include low parity and a family historyof ovarian cancer. Less than 0.1% of women are affected by hereditaryovarian cancer syndrome, but these women may face a 40% lifetime risk ofdeveloping ovarian cancer.

Potential screening tests for ovarian cancer include the bimanual pelvicexamination, the Papanicolaou (Pap) smear, tumor markers, and ultrasoundimaging. The pelvic examination, which can detect a variety ofgynecologic disorders, is of unknown sensitivity in detecting ovariancancer. Although pelvic examinations can occasionally detect ovariancancer, small, early-stage ovarian tumors are often not detected bypalpation due to the deep anatomic location of the ovary. Thus, ovariancancers detected by pelvic examination are generally advanced andassociated with poor survival. The pelvic examination may also producefalse positives when benign adnexal masses (e.g., functional cysts) arefound. The Pap smear may occasionally reveal malignant ovarian cells butit is not considered a valid screening test for ovarian carcinoma.Ultrasound imaging has also been evaluated as a screening test forovarian cancer, since it is able to estimate ovarian size, detect massesas small as 1 cm, and distinguish solid lesions from cysts.

Serum tumor markers are often elevated in women with ovarian cancer.Examples of these markers include carcinoembryonic antigen, ovariancystadenocarcinoma antigen, lipid-associated sialic acid, NB/70K, TAG72.3, CA15-3, and CA-125. respectively. Evidence is limited on whethertumor markers become elevated early enough in the natural history ofoccult ovarian cancer to provide adequate sensitivity for screening.Tumor markers may have limited specificity. It has been reported thatCA-125 is elevated in 1% of healthy women, 6-40% of women with benignmasses (e.g., uterine fibroids, endometriosis, pancreatic pseudocyst,pulmonary hamartoma), and 29% of women with nongynecologic cancers(e.g., pancreas, stomach, colon, breast). Prospective studies involvingasymptomatic women are needed, however, to provide definitive data onthe performance characteristics of serum tests when used as screeningtests.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention is directed to amonoclonal antibody or binding fragment thereof which specifically bindsto antigens sharing a common epitope present on the surface of humanmyeloma cells and ovarian cancer cells. The antigen on multiple myelomacells is a single, glycosylated polypeptide with a molecular weight ofabout 78 kDa to about 120 kDa as determined by SDS PAGE under reducingconditions. The antigen on ovarian cancer cells is a single,glycosylated polypeptide with a molecular weight of about 76 kDa toabout 213 kDa as determined by SDS PAGE under reducing conditions. Theantigens are absent from human peripheral blood mononuclear cells,absent from human B cells, and absent from human chronic myelogenicleukemia cells. A non-limiting example of the monoclonal antibody isthat produced by the hybridoma cell line deposited at the American TypeCulture Collection having accession No. PTA-450. Furthermore, theantigens are not present on cells from a breast cancer tumor, notpresent on a prostate cancer cell line, not present on a neuroblastomacells line, and not present on a cervical cancer cell line. They arealso not found on an Epstein-Barr virus-transformed B cell tumor.

The present invention is further directed to antibodies that are capableof binding to the same antigenic determinant as does the monoclonalantibody produced by the hybridoma cell line deposited at the AmericanType Culture Collection having accession No. PTA-450; binding fragmentsof the hybridoma cell line deposited at the American Type CultureCollection having accession No. PTA-450; and to binding fragments of amonoclonal antibody capable of binding to the same antigenic determinantas does the monoclonal antibody produced by the hybridoma cell linedeposited at the American Type Culture Collection having accession No.PTA-450.

Such monoclonals or fragments may be human, or may be of other mammalianspecies such as rodent, hybrids thereof, chimeric antibodies, and thelike. Binding fragments of the monoclonal antibodies of the presentinvention include but are not limited to F(ab′)₂, Fab′, Fv, Fd′, or Fdfragments.

In another aspect, the present invention is directed to a cell lineproduced by a hybridoma technique, which produces a monoclonal antibodywhich specifically binds to surface antigens of human myeloma cells andof ovarian cancer cells. The antigen on multiple myeloma cells is asingle, glycosylated polypeptide with a molecular weight of about 78 kDato about 120 kDa as determined by SDS PAGE under reducing conditions.The antigen on ovarian cancer cells is a single, glycosylatedpolypeptide with a molecular weight of about 76 kDa to about 213 kDa asdetermined by SDS PAGE under reducing conditions. The antigens areabsent from human peripheral blood mononuclear cells, absent from humanB cells, and absent from human chronic myelogenic leukemia cells. Anon-limiting example of the monoclonal antibody is that produced by thehybridoma cell line deposited at the American Type Culture Collectionhaving accession No. PTA-450. Furthermore, the antigens are not presenton cells from a breast cancer tumor, not present on a prostate cancercell line, not present on a neuroblastoma cells line, and not present ona cervical cancer cell line. They are also not found on an Epstein-Barrvirus-transformed B cell tumor.

A further aspect of the present invention is the hybridoma cell linedeposited at the American Type Culture Collection having accession No.PTA-450.

In another broad aspect of the present invention, an isolated surfaceantigen of human multiple myeloma cells is described, the antigen beinga single, glycosylated polypeptide with a molecular weight of about 78kDa to about 120 kDa as determined by SDS PAGE under reducingconditions; the antigen being absent from human peripheral bloodmononuclear cells, absent from human B cells, and absent from humanacute myelogenic leukemia cells. The antigen is not present on cellsfrom a breast cancer tumor, not present on a prostate cancer cell line,not present on a neuroblastoma cells line, and not present on a cervicalcancer cell line. It is also not found on an Epstein-Barrvirus-transformed B cell tumor. The isolated multiple myeloma surfaceantigen binds to a monoclonal antibody produced by the hybridoma cellline deposited at the American Type Culture Collection having accessionNo. PTA-450.

In another broad aspect of the present invention, an isolated surfaceantigen of human ovarian cancer cells is described, the antigen being asingle, glycosylated polypeptide with a molecular weight of about 76 kDato about 213 kDa as determined by SDS PAGE under reducing conditions;the antigen being absent from human peripheral blood mononuclear cells,absent from human B cells, and absent from human acute myelogenicleukemia cells. The antigen is not present on cells from a breast cancertumor, not present on a prostate cancer cell line, not present on aneuroblastoma cells line, and not present on a cervical cancer cellline. It is also not found on an Epstein-Barr virus-transformed B celltumor. The isolated ovarian cancer surface antigen binds to a monoclonalantibody produced by the hybridoma cell line deposited at the AmericanType Culture Collection having accession No. PTA-450.

The present invention is also directed to methods for inhibiting orkilling myeloma cells in a patient by administering the monoclonalantibody or binding fragment as described above under conditionssufficient for the binding of the monoclonal antibody or bindingfragment to the myeloma cells to cause inhibiting or killing of thecancer cells by the immune cells of the patient. In another aspect, amethod for inhibiting or killing myeloma cells in a patient is providedby administering the monoclonal antibody or binding fragment asdescribed above which is conjugated with a cytotoxic moiety, underconditions sufficient for the binding of the monoclonal antibody orbinding fragment to the cancer cells to cause inhibiting or killing ofthe cells. The cytotoxic moiety may be, by way of non-limiting example,a chemotherapeutic agent, a photo-activated toxin or a radioactiveagent.

In still another aspect of the invention, the above-mentioned conjugateof the monoclonal antibody or binding fragment described herein and acytotoxic moiety may be used in vitro to inhibit or kill myeloma cellsin a cellular sample, such as a bone marrow sample.

The invention is also directed to anti-idiotypic antibodies which mirrorthe binding site of the monoclonal antibody of the invention, and arcspecific to the myeloma and ovarian cancer conformational epitoperecognized by the antibody of the invention. The invention is furtherdirected to the use of the aforementioned anti-idiotypic antibodies forthe treatment of MM or ovarian cancer by active immunization.

In a further aspect of the invention, a method is provided for removingmyeloma cells from an isolated cellular sample, such as, but not limitedto, bone marrow cells, by exposing the cellular sample to a solid matrixon which the monoclonal antibody or binding fragment described above isbound under conditions wherein the myeloma cells adhere to themonoclonal antibody or binding fragment, and isolating a cellularfraction of said cellular sample which does not bind to the matrix. Thismethod may be used, for example, in the removal of myeloma cells from abone marrow sample for autologous bone marrow transplant.

The invention is also directed to the monoclonal antibody or bindingfragment as described above bound to a solid support.

In yet another aspect of the invention, a method is provided forlocalizing myeloma cells or tumors, or ovarian cancer cells or tumors,in a patient by administering the monoclonal antibody or bindingfragment described above, allowing the monoclonal antibody or bindingfragment thereof to bind to the cancer cells within said patient, anddetermining the location of the monoclonal antibody or binding fragmentthereof within the patient. In another related aspect, the monoclonalantibody or binding fragment is detectably labeled, for example, with aradionuclide.

The present invention is further directed to methods for inhibiting orkilling ovarian cancer cells in a patient by administering themonoclonal antibody or binding fragment as described above underconditions sufficient for the binding of the monoclonal antibody orbinding fragment to the ovarian cancer cells to cause inhibiting orkilling of the ovarian cancer cells by immune cells of the patient. Inanother aspect, a method for inhibiting or killing ovarian cancer cellsin a patient is provided by administering the monoclonal antibody orbinding fragment as described above which is conjugated with a cytotoxicmoiety, under conditions sufficient for the binding of the monoclonalantibody or binding fragment to ovarian cancer cells to cause inhibitingor killing of the ovarian cancer cells. The cytotoxic moiety may be, byway of non-limiting example, a chemotherapeutic agent, a photo-activatedtoxin or a radioactive agent.

In yet another aspect of the invention a method is provided forlocalizing ovarian cancer cells in a patient by administering themonoclonal antibody or binding fragment described above, allowing themonoclonal antibody or binding fragment thereof to bind to ovariancancer cells within said patient, and determining the location of saidmonoclonal antibody or binding fragment thereof within said patient. Inanother related aspect, the monoclonal antibody or binding fragment isdetectably labeled, for example with a radionuclide.

It is a further aspect of the invention to permit the detection of thecell surface glycoproteins described herein in a sample of bodily fluid,to aid in the diagnosis of multiple myeloma, ovarian cancer, or othercancer cells expressing a glycoprotein with the epitope recognized bythe antibodies herein by the detection of the glycoprotein antigen shedfrom cancer cells into the bodily fluid, such as blood. Furthermore, thestate of the disease may be monitored and the effectiveness ofanticancer therapies monitored by determining the level or changes overtime of the level of shed surface glycoprotein in a bodily fluid such asblood.

In still yet another aspect, the invention is directed to pharmaceuticalcompositions comprising a monoclonal antibody or binding fragment asdescribed above, and a pharmaceutically-acceptable carrier or diluent.

In another aspect, the present invention is directed to a monoclonalantibody or binding fragment as described above labeled with adetectable moiety, such as, by way of non-limiting examples, afluorophore, a chromophore, a radionuclide, or an enzyme.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a first screen of B cell hybridomas generated from miceimmunized with a pool of three human plasmacytoma cells as compared withtheir binding to human myelogenic leukemia cell line (K562, serving as acontrol).

FIG. 2 presents the result of selected hybridomas for the second screen.

FIG. 3 depicts the net binding values obtained for the first screen incomparison with the second screen.

FIG. 4 presents the results of cell surface staining using a panel ofmonoclonal antibodies analyzed by flow cytometry.

FIG. 5 represents further evaluation of the selected monoclonalantibodies using Western blot method, using membrane proteins extractedfrom 5 human myeloma cell lines tested individually, and controls,fractionated on SDS-PAGE.

FIG. 6 presents a similar experiment as in FIG. 5, using a cellularELISA method.

FIG. 7 shows an SDS-PAGE gel of concentrated culture fluid from multiplemyeloma cells grown in serum-free medium for 5 days, blotted and probedwith an antibody of the invention.

FIG. 8 depicts an SDS-PAGE of cell lysates from three ovarian cancertumors from 3 patients which were digested with trypsin and homogenized.The gel was blotted and probed with an antibody of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Identification of unique cancer antigens enables the design of selectiveimmunotherapy for neoplastic diseases. The capacity to utilize adeterminant exclusively expressed by cancer cells, and which arc devoidin normal tissues, ensures the targeting and elimination of theneoplastic cells while insulating the function of normal cells. Althoughthe last decades have witnessed great activity and significant successin the search for novel cancer antigens for various neoplastic diseases,for many malignancies the cancer-specific antigens have not yet beendefined. The majority of cancer antigens are self-antigens that arcderived from and expressed by the normal cells. Frequently, the cancerantigen is identical to the normal antigen though it is expressed athigher levels or endowed with a negligible mutation insufficient for itsdistinction from the self-antigen. One of the escape mechanisms ofmalignant cells from the immune system is their similarity to theirnormal counterpart resulting in their low visibility by the immunesystem.

The inventors herein have identified new surface glycoprotein antigenspresent on human myeloma cells and human ovarian cancer tumor cells butabsent from normal cells and from leukemic cells that presents a targetfor therapeutic intervention in myeloma and ovarian cancer as well asfor diagnostic and cell purification purposes. These antigens share atleast one common epitope. The inventors used a technique known ascontrasting immunization for obtaining monoclonal antibodies to theantigen and for the identification of the novel cancer antigen describedherein. As will be seen in the examples below, two divergent immunogensprovided at different locations were used. The dual immunizationpolarizes the migration of the distinct populations of immune cells todiscrete draining lymph nodes. In an example herein, a mixture of humanmyeloma cells was used as the immunogen to obtain murine monoclonalantibodies to a myeloma cell surface antigen. Control cells, in thisexample, a related, myelogenic leukemic cell line, were used to polarizethe immune response, to effectively delete undesired cells from thelymph nodes near the site of immunization with the desired antigen. Theimmune cells extracted from the draining lymph nodes close to theimmunization site with the desired neoplasms were immortalized by fusionwith murine myeloma cells. The antipodal draining lymph nodes werepopulated with immune cells specific to the undesired (control)immunogens.

By use of the foregoing protocol, a series of monoclonal antibodies wereprepared which were found to that specifically bind specifically toantigens on the surface of human myeloma cells and on ovarian cancercells. These antigens share at least one epitope. The antigens arefurther characterized in that the antigen on multiple myeloma cells is asingle, glycosylated polypeptide with a molecular weight of about 78 kDato about 120 kDa as determined by SDS PAGE under reducing conditions;and it is absent from human peripheral blood mononuclear cells, absentfrom human B cells, and absent from human B cell myelogenic leukemiacells. The antigen on ovarian cancer cells is a single, glycosylatedpolypeptide with a molecular weight of about 76 kDa to about 213 kDa asdetermined by SDS PAGE under reducing conditions; and it is absent fromhuman peripheral blood mononuclear cells, absent from human B cells, andabsent from human B cell myelogenic leukemia cells. An antigenrecognized by the antibody of the invention is also present on livercancer cells; thus, the liver cancer cell surface antigen shares thesame at least one epitope as the myeloma and ovarian cancer surfaceglycoprotein. An example of a hybridoma that produces a monoclonalantibody which recognizes these antigens has been deposited at theAmerican Type Culture Collection, and accorded accession No. PTA-450.

The aforementioned antigens were not present on cells from a breastcancer tumor, not on a prostate cancer cell line, not on a neuroblastomacells line, and not on a cervical cancer cell line. They were also notfound on an Epstein-Barr virus-transformed B cell tumor.

The present invention is directed to monoclonal antibodies and bindingfragments thereof which recognize the aforementioned myeloma cell andovarian cancer cell surface glycoproteins. Thus, the present inventionembraces the deposited monoclonal antibody described above andmonoclonal antibodies as well as their binding fragments with thebinding specificity for the aforementioned antigens. Such antibodyfragments capable of binding the aforementioned antigens, includes butis not limited to F(ab′)₂ fragments, Fab′ fragments, Fv fragments, Fd′fragments, or Fd fragments. Antibodies may be human mammalian such asmouse, and hybrid or chimeric antibodies. These fragments and means forpreparing then from antibodies are known to one of skill in the art.

Thus, the monoclonal antibodies and binding fragments may becharacterized as those which are 1) produced from the hybridoma cellline deposited at the American Type Culture Collection having accessionNo. PTA-450; 2) antibodies that are capable of binding to the sameantigenic determinant as does the monoclonal antibody produced by thehybridoma cell line deposited at the American Type Culture Collectionhaving accession No. PTA-450; 3) binding fragments of the hybridoma cellline deposited at the American Type Culture Collection having accessionNo. PTA-450; or 4) binding fragments of a monoclonal antibody capable ofbinding to the same antigenic determinant as does the monoclonalantibody produced by the hybridoma cell line deposited at the AmericanType Culture Collection having accession No. PTA-450.

Thus, the aforementioned monoclonal antibodies and binding fragmentsrecognize a common epitope of cell surface glycoproteins present onhuman myeloma cells and on human ovarian cancer cells, but are absentfrom human peripheral blood mononuclear cells, absent from human Bcells, and absent from human B cell myelogenic leukemia cells, andfurther, the cell surface glycoprotein on myeloma cells is a singlepolypeptide with a molecular weight of about 78 kDa to about 120 kDa asdetermined by SDS PAGE under reducing conditions. The cell surfaceglycoprotein on ovarian cancer cells is a single polypeptide with amolecular weight of about 76 kDa to about 213 kDa as determined by SDSPAGE under reducing conditions. As the myeloma, ovarian cancer, andliver cancer cell surface glycoproteins share a common epitoperecognized by the antibodies of the invention, such antibodies may beused therapeutically and diagnostically for these conditions. Asmentioned above, the antigen is also present on the surface of livercancer cells, but is not present on cells from a breast cancer tumor,not present on a prostate cancer cell line, not present on aneuroblastoma cells line, and not present on a cervical cancer cellline. It is also not found on an Epstein-Barr virus-transformed B celltumor.

The present invention is also directed to hybridoma cell lines whichproduce a monoclonal antibody which specifically binds to the surfaceantigens of human myeloma cells and ovarian cancer cells described andcharacterized herein. These antigens have a shared region or an epitopecontained in the cell surface glycoproteins of these neoplasms. Themethods for the preparation of such hydridomas are known to the skilledartisan. The contrasting immunization procedure described herein is butone example of various means for obtaining the desired antibodies. Forpreparation of monoclonal antibodies directed toward the surfaceglycoprotein antigens described herein, any technique that provides forthe production of antibody molecules by continuous cell lines in culturemay be used. These include but are not limited to the hybridomatechnique originally developed by Kohler and Milstein [Nature256:495-497 (1975)], as well as the trioma technique, the human B-cellhybridoma technique [Kozbor et al., Immunology Today 4:72 1983); Cote etal., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030 (1983)], and theEBV-hybridoma technique to produce human monoclonal antibodies [Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96 (1985)]. In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizing thetechnology described in WO9802545. In fact, according to the invention,techniques developed for the production of “chimeric antibodies.” Suchhuman or humanized chimeric antibodies are preferred for use in therapyof human diseases or disorders as described infra, since the human orhumanized antibodies are much less likely than xenogenic antibodies toinduce an immune response, in particular an allergic response,themselves.

According to the invention, techniques described for the production ofsingle chain antibodies [U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778] can be adapted to produce myelomasurface antigen-specific single chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries [Huse et al., Science246:1275-1281 (1989)] to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity, or itsderivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

As mentioned above, the present invention is also directed to theisolated surface antigens of human myeloma cells and human ovariancancer cells, the former antigen being characterized in that it is asingle, glycosylated polypeptide with a molecular weight of about 78 kDato about 120 kDa as determined by SDS PAGE under reducing conditions;the latter with a molecular weight of about 76 kDa to about 213 kDa.These antigens are absent from human peripheral blood mononuclear cells,absent from human B cells, and absent from human B cell myelogenicleukemia cells. The antigens are also absent from breast cancer, asdetermined using fresh tumor tissue; absent from prostate cancer using aprostate cancer cell line; absent from a neuroblastoma using aneuroblastoma cell line, and absent from cervical cancer using acervical cancer cell line. The glycoproteins have been found to bepresent on the surface of cells from a freshly-isolated liver cancertumor; the foregoing methods are also applicable to the therapy anddiagnosis of liver cancer. The isolated surface antigens are furthercharacterized in that they binds to the monoclonal antibody produced bythe hybridoma cell line deposited at the American Type CultureCollection having accession No. PTA-450.

The monoclonal antibody MA69 reacts consistently with a single chainglycoprotein with a molecular weight of 78-120 kDa on multiple myelomacells. On ovarian carcinomas, however. MA69 recognizes one or moreglycoproteins ranging in size from 76 to 213 kDa. These results implythat MA69 reacts with 2 distinct molecules expressed on MM and ovariancancer through the recognition of a shared region or an epitopecontained in the cell surface glycoproteins of these neoplasms. Thisepitope is uniquely expressed on ovarian and MM malignancies and was notfound on the cell surface of a panel of human tumors such as lungcancer, cervical cancer, neuroblastoma, breast cancer, prostate cancer,leukemia and lymphomas. Thus, the invention is also directed to cellsurface glycoproteins generally which comprise an epitope recognized bythe antibodies of the invention. As noted above, cell surfaceglycoproteins comprising this epitope are absent from the various normaland cancer cells tested, as listed above.

The present invention is also directed to therapeutic methods for thetreatment of myeloma and related dysproliferative diseases in humans,including multiple myeloma, as well as ovarian cancer, using theantibodies of the present invention. The therapeutic as well asdiagnostic uses described herein embrace primary tumors as well asmetastases. For example, a method for inhibiting or killing myelomacells or ovarian cancer cells in a patient may be carried out byadministering to the patient, in a single dose or in successive doses,the monoclonal antibody or binding fragment as described above, underconditions sufficient for the binding of the monoclonal antibody orbinding fragment to tumor cells in the patient. Binding of antibodies tothe tumor cells induces the inhibiting or killing of the tumor cells byimmune cells in the patient. The aforementioned therapy may beaccompanied by other treatments directed to the tumor cells, such aschemotherapy, radiation, etc., as well as to adjunctive therapies toenhance the immune system attack on the opsonized tumor cells followingthe procedure described above. For example, the patient may beco-administered with a growth factor such as erythropoietin and/orGM-CSF for stimulating the white blood cells and supporting theimmunocompetence status of the patient. Furthermore chimeric or otherrecombinant antibodies of the invention, may be used therapeutically.For example, a fusion protein comprising at least the antigen-bindingregion of the antibody of the invention joined to at least afunctionally active portion of a second protein having anti-tumoreffects, e.g., a lymphokine or oncostatin, may be used to treat humantumor in vivo. In addition, a chimeric antibody wherein theantigen-binding site is joined to human Fe region, e.g., IgG1, may beused to promote antibody-dependent mediated cytotoxicity orcomplement-mediated cytotoxicity. Furthermore, recombinant techniquesknown in the art can be used to construct bispecific antibodies whereinone of the binding specificities is that of the antibody of theinvention (See. e.g., U.S. Pat. No. 4,474,893).

Of course, other dysproliferative diseases in which the glycoproteinantigens of the invention are present on the cell surface are treatableby the methods described herein.

The foregoing method uses the antibodies or binding fragments withoutmodification, relying on the binding of the antibodies to the surface ofthe myeloma or ovarian cancer cells in situ to stimulate immune attackthereon. In another aspect of the therapeutic methods, theaforementioned method may be carried out using the monoclonal antibodiesor binding fragments to which a cytotoxic agent is bound. Binding of thecytotoxic antibodies or binding fragments to the tumor cells inhibitsthe growth of or kills the cells. By way of non-limiting examples,suitable cytotoxic agents may be chemotherapeutic agent, aphoto-activated toxin or a radioactive agent. For example, cytotoxicagents such as ricin A chain, abrin A chain, modeccin A chain, gelonin,melphalan, bleomycin, adriamycin, daunomycin, or pokeweed antiviralproteins (PAP, PAPII, PAP-S). Those skilled in the art will realize thatthere are numerous radioisotopes and chemocytotoxic agents that can becoupled to tumor specific antibodies by well known techniques, anddelivered to specifically destroy tumor tissue. See e.g. Blattler et al.U.S. Pat. No. 4,542,225. Examples of photo-activated toxins includedihydropyridine- and omega-conotoxin (Schmidt et al. J Biol Chem, 1991,266:27, 18025-33). Examples of imaging and cytotoxic reagents that canbe used include ¹²⁵I, ¹¹¹In, ¹²³I, ^(99m)Tc, ³²P, ³H, and ¹⁴C,fluorescent labels such as fluorescein and rhodamine, andchemiluminescers such as luciferin. The antibody can be labeled withsuch reagents using techniques known in the art. For example, see Wenzeland Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, N. Y.(1983) for techniques relating to the radiolabeling of antibodies (seealso, Colcer et al., “Use of Monoclonal Antibodies AsRadiopharmaceuticals For The Localization Of Human Carcinoma XenograftsIn Nude Mice” Meth. Enzymol., 121: 802-16 (1986), “Order, Analysis,Results and Future Prospective of the Therapeutic Use of RadiolabeledAntibody in Cancer Therapy”, in Monoclonal Antibodies for CancerDetection and Therapy, Baldwin et al. (eds), pp. 303-16 (Academic Press1985).

Other covalent and non-covalent modifications of the antibodies orfragments are embraced herein, including agents, which areco-administered or administered after the antibody or fragments, toinduce inhibition or killing of the cells to which the antibody orfragment has previously bound.

Anti-idiotypic monoclonal antibodies to the antibodies of the inventionmay be used therapeutically in active tumor immunization and tumortherapy (see, e.g., Hellstrom et al., “Anti Idiotypes” in CovalentlyModified Antigens and Antibodies in Diagnosis and Therapy, supra at pp.35-41).

In the area of multiple myeloma, the antibodies or fragments of thepresent invention have further utility in the preparation of cellularsamples from which myeloma cells have been removed. This is particularlyimportant in autologous bone marrow transplants, wherein a sample ofbone marrow is harvested from a cancer patient prior to the patientundergoing high-dose chemotherapy. The goal of the high dosechemotherapy is to destroy the cancer cells, which also results in thedepletion of bone marrow cells. Following such treatment the harvestedbone marrow cells are reintroduced to the patient. In myeloma andrelated diseases, the harvested bone marrow is contaminated with myelomacells; reintroduction of untreated bone marrow will simply reintroducethe disease. Previous methods to prevent reintroduction of cancer cellshave included treatment of the bone marrow sample with chemotherapeuticagents and other anti-neoplastic agents in vitro; other methods includepurging the sample of cancer cells. In a further practice of the presentinvention, the monoclonal antibodies and fragments described herein maybe used to remove myeloma cells from a bone marrow sample beforereintroduction to the patient. In one non-limiting example, themonoclonal antibody or binding fragments are attached to a matrix suchas beads. This may be accomplished by any of several well-known methodsfor preparing an affinity matrix comprising antibodies or bindingfragments. The bone marrow sample is exposed to the matrix, such as bypassage of the cells over a column containing the matrix, underconditions to promote the binding of the myeloma cells in the samplethrough antigen/antibody interactions with the antibody or bindingfragments on the matrix. The myeloma cells in the sample adhere to thematrix; the column effluent or non-adherent cellular population isdepleted of myeloma cells. The effectiveness of the procedure may bemonitored by examining the cells for residual myeloma cells, such as byusing a detectably-labeled antibody as described below. The proceduremay be repeated or modified to increase effectiveness. This purgingprocedure [see, e.g., Ramsay et al., J. Clin. Immunol., 8(2): 81-88(1988)] may be performed together with other methods for removing orkilling cancer cells, including but not limited to exposing the purifiedbone marrow cells to chemotherapeutic agents. Such chemotherapeuticagents include the use of the antibodies or binding fragments of thepresent invention conjugated to a cytotoxic agent, as described abovefor in-vivo therapeutic treatment. Thus, conjugates of the antibodies orfragments of the invention with cytotoxic agents may be used for theex-vivo killing of tumor cells in a cellular sample. These methods mayinclude additionally exposing the cells to cytokines (GM-CSF, IL-6),cytokine receptors (such ad IL-6-receptor), mitogens (such as PWM-Pokeweed mitogen) or adhesion molecules (such as CD40 ligand) in order tostimulate the myeloma cells to rapidly differentiate and therebyupregulate expression of cancer-specific antigens on their cell surface.These treatment modalities are aimed to render the myeloma cellsvulnerable to the in-vitro mediated cytotoxicity instigated byincubation with the monoclonal antibody.

In another aspect of the therapeutic methods of the present invention,the antibodies or binding fragments conjugated with cytotoxic agents,such as chemotherapeutic agents, a photo-activatable toxin, or aradionuclide, may be used in vitro or ex vivo to inhibit or kill myelomacells from a bone marrow sample, in the absence of the purging techniquedescribed above. The treatment of the sample with the cytotoxicantibodies or fragments may be combined with other methods to killcancer cells to increase the effectiveness of a bone marrow transplantparticularly an autologous bone marrow transplant, by removing cancelcells from the tissue to be transplanted. These methods may includeadditionally exposing the cells to cytokines, etc. Thus, a method isdescribed herein for removing myeloma cells from a isolated cellularsample comprising the steps of exposing the cellular sample to a solidmatrix on which said monoclonal antibody or binding fragment asdescribed herein is bound under conditions wherein the myeloma cellsadhere to the monoclonal antibody or binding fragment thereof, andisolating a cellular fraction of the cellular sample which does not bindto the matrix. By way of non-limiting example, bone marrow cells areused, particularly for a transplant, and preferably, an autologous bonemarrow transplant.

In a further aspect of the present invention, compositions comprisingthe monoclonal antibody or binding fragment as described herein bound toa solid support. A solid support for use in the present invention willbe inert to the reaction conditions for binding. A solid phase supportfor use in the present invention must have reactive groups or activatedgroups in order to attach the monoclonal antibody or binding partner. Inanother embodiment, the solid phase support may be a usefulchromatographic support, such as the carbohydrate polymers SEPHAROSE(R),SEPHADEX(R), or agarose. As used herein, a solid phase support is notlimited to a specific type of support. Rather a large number of supportsare available and are known to one of ordinary skill in the art. Solidphase supports include silica gels, resins, derivatized plastic films,glass beads, cotton, plastic beads, alumina gels, magnetic beads,membranes (including but not limited to nitrocellulose, cellulose,nylon, and glass wool), plastic and glass dishes or wells, etc.

The present invention is also directed to diagnostic and imaging methodsfor multiple myeloma and ovarian cancer using the monoclonal antibodiesand binding fragments as described hereinabove. Other cancers bearingthe surface antigen of the invention are also amenable to thesediagnostic procedures. The method involves administration or infusion ofmonoclonal antibodies or binding fragments as described herein, with orwithout conjugation to a detectable moiety, such as a radionuclide.After administration, the antibody or fragment binds to the tumor cells,after which the location of the antibodies or fragments is detected. Fordetectably labeled antibodies or fragments, such as those labeled with aradionuclide, imaging instrumentation may be used to identify thelocation within the body of the agent. For use of unlabeled antibodiesor fragments, a second, detectable reagent may be administered whichlocates the antibodies or fragments, and thus may be suitably detected.These methods have been used for other antibodies and the skilledartisan will be amply aware of these various methods for imaging thelocation of antibodies or fragments within the body.

In the case of ovarian cancer as well as other cancers expressing theantigens described herein, the invention is further directed to thediagnosis of cancer by the identification and measurement of shed cellsurface glycoprotein in bodily fluids such as blood. As ovarian canceris a particularly difficult cancer to diagnose in its early stage, thusthwarting the opportunity for early treatment, methods for earlydiagnosis are particularly needed. Measurement of shed surfaceglycoprotein in a whole blood sample, by use of an antibody of theinvention or another method, provides such early diagnosis and theopportunity for treatment. Such treatment may comprise the foregoingantibody-based therapy, in combination with other agents, or agentswithout the antibody of the invention.

Furthermore, the level of shed ovarian cancer antigen measured in bloodor other bodily fluids provides a means for monitoring the course ofovarian cancer therapy, including surgery, chemotherapy, radiationtherapy, and the therapeutic methods of the present invention. Bycorrelating the level of shed antigen with the severity of disease, thelevel of shed antigen can be used to indicate successful removal of theprimary tumor and/or metastases, and the effectiveness of othertherapies over time. A decrease in the level over time indicates areduced tumor burden in the patient; in contrast, no change or anincrease indicates ineffectiveness of therapy or the continues growth ofthe tumor.

The present invention is also directed to pharmaceutical compositionscomprising a monoclonal antibody or binding fragment which specificallybinds to an antigen on the surface of a human myeloma cell, the antigenbeing further characterized as described hereinabove, together with apharmaceutically-acceptable carrier or diluent. The invention is furtherdirected to pharmaceutical compositions comprising a monoclonal antibodyor binding fragment including the monoclonal antibody produced from thehybridoma cell line deposited at the American Type Culture Collectionhaving accession No. PTA-450; antibodies that are capable of binding tothe same antigenic determinant as does the monoclonal antibody producedby the hybridoma cell line deposited at the American Type CultureCollection having accession No. PTA-450; binding fragments of thehybridoma cell line deposited at the American Type Culture Collectionhaving accession No. PTA-450; and binding fragments of a monoclonalantibody capable of binding to the same antigenic determinant as doesthe monoclonal antibody produced by the hybridoma cell line deposited atthe American Type Culture Collection having accession No. PTA-450; and apharmaceutically-acceptable carrier or diluent. Antibody fragmentsinclude but are not limited to F(ab′)₂ fragments, Fab′ fragments, Fvfragments, Fd′ fragments, or Fd fragments.

A pharmaceutical composition includes a pharmaceutically acceptablecarrier or diluent. Preferably, the antibodies or binding fragmentsthereof are delivered parenterally, such as by intravenousadministration. Suitable buffers, carriers, and other components knownto the art will be used in formulating a composition comprising theantibody or fragments for suitable shelf-life and compatibility withadministration. These substances may include ancillary agents such asbuffering agents and protein stabilizing agents (e.g., polysaccharides).

The antibody of the invention is also useful for diagnosticapplications, both in vitro and in vivo, for the detection of humanmultiple myeloma and ovarian cancer cells that possess the antigen forwhich the antibodies are specific. In vitro diagnostic methods includeimmunohistological detection of tumor cells (e.g., on human tissue,cells for excised tumor specimens) or serological detection of tumorassociated antigens (e.g., in blood samples or other biological fluids).Immunohistochemical techniques involve staining a biological specimensuch as tissue specimen with the antibody of the invention and thendetecting the presence on the specimen of such antibody-antigencomplexed to its antigen. The formation of such antibody-antigencomplexes with the specimen indicates the presence of multiple myelomacells in the tissue. Detection of the antibody on the specimen can beaccomplished using techniques known in the art such as immunoenzymatictechniques, e.g., immunoperoxidase staining technique or theavidin-biotin technique, or immunofluorescence techniques (see, e.g.,Ciocca et al., “Immunohistochemical Techniques Using MonoclonalAntibodies”, Meth. Enzymol, 121:562-79 (1986), and Kimball (ed)Introduction to Immunology (2^(nd) Ed) pp. 113-117 (Macmillan Pub. Co.1986). Serologic diagnostic techniques involve the detection andquantitation of tumor-associated antigens that have been secreted or“shed” into the serum or other biological fluids of patients thought tobe suffering from multiple myeloma. Such antigens can be detected in thebody fluids using techniques known in the art such as radioimmunoassay(RIA) or enzyme-linked immunoabsorbant assays (ELISA) wherein antibodyreactive with the “shed” antigen is used to detect the presence of theantigen in a fluid sample (see, e.g., Uotila et al. “Two-Site SandwichELISA With Monoclonal Antibodies to Human AFP”, J. Immunolo. Methods,42:11 (1981) and Allum et al., supra at pp 48-51). Detection of the shedovarian cancer antigen is described above.

As mentioned above, the antibodies of the invention are useful for themeasurement of shed ovarian cancer cell antigen in bodily fluids such aswhole blood, for the diagnosis of cancer and the monitoring of theeffectiveness of therapies.

In yet a further aspect of the invention, monoclonal antibodies orbinding fragments to the myeloma surface glycoprotein and the ovariancancer glycoprotein are provided labeled with a detectable moiety, suchthat they may be used to diagnose or identify cells having theaforementioned antigen. Non-limiting examples of such labels includefluorophores such as fluorescein isothiocyanate; chromophores,radionuclides, or enzymes. Such labeled antibodies or binding fragmentsmay be used for the histological localization of the antigen, ELISA,cell sorting, and other immunological techniques for detecting orquantitating the antigen, and cells bearing the antigen, for example. Asnoted above, one use of such labeled antibodies or fragments is indetermining the effectiveness of myeloma cell depletion from bone marrowtissue prior to transplant, particularly autologous bone marrowtransplant.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which arc provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1 Preparation and Screening of Hybridomas

1. Sources of cells. Human myeloma cell lines (U266, OPM, RPMI1860, KR12and NCI H929), and chronic myelogenic leukemic cell line (K562) werepurchased from the American Type Culture Collection (ATCC). Fresh, humanovarian cancer, breast cancer, and liver cancer specimens were used.Cell lines of prostate cancer, LnCap (ATCC); neuroblastoma cell line,NCI H2106 (ATCC); and a cervical cancer, Caski (ATCC) were alsoevaluated, as well as an EBV-transformed B cell tumor, Namalwa (ATCC).

2. Immunization. Mice were immunized with a pool of plasmacytoma cells,U266, RPMI1860 and OPM (5×10⁶ total in 50 μl containing Ribi adjuvant[50%]), in the left footpad and with K562 cells (5×10⁶ total in 50 μlcontaining Ribi adjuvant [50%]) in the right footpad. The immunizationwas repeated after 14 days. The left popliteal lymph node was removedand the extracted cells were fused 3 days after the second immunization.

3. Generation of B cell hybridomas. Monoclonal antibodies specific tomultiple myeloma cells are produced by conventional methods. Popliteal(left) lymph node cells from immunized mice were fused with a mousemyeloma cell line (Sp2/o) in the presence of poly ethylene glycol (PEG),thus forming hybridomas which are capable of producing monoclonalantibodies which specifically bind to human plasmacytoma cells.

4. Cellular ELISA—Flow cytometry analysis. Various human tumor celllines grown in in-vitro culture were washed, and stained with a panel ofmonoclonal antibodies selected based on cellular ELISA screen. After 30minutes of incubation on ice, the cells were washed and incubated withRabbit anti mouse gamma monoclonal antibody conjugated with fluoresceinisothiocyanate (FITC). Mean intensity of the fluorescence weredetermined by flow cytometry using the FACScaliber (Becton andDickinson). Histograms plotting the intensity of the staining incorrelation with cell count demonstrated the specificity of monoclonalantibody 69 (MA69) to human plasmacytoma cells.

5. Western blot analysis. SDS-PAGE gels were prepared from stocksolutions of 30% acylamide/0.8% bisacrylamide, TRIS-HCl/SDS, pH 8.8 wasadded, sterile distilled H₂O, 10% (w/v) ammonium persulfate was added,and TEMED, following standard procedures. A stacking gel was included ifthe samples were greater than 10 μl. Surface membrane proteins fromcells were prepared for electrophoresis by the following protocol: Cellsfrom in vitro culture were collected and washed. The cells were lysedfollowing 3 repeated cycles of freeze—thaw (−80° C. and 37° C.). Thelysates were stored at −20° C. until use. Membranes were prepared fromcell lysates following a 30 min. centrifugation at 2500 rpm. Thesupernatant consisting of cytosolic protein and membranes was furtherseparated by centrifugation at 40,000 rpm using ultra-centrifuges. Thepellet containing the membrane fraction was collected and stored at −20°C.

Proteins were separated at 150 V for about 1.5 hours at 4° C. Afterseparation, the proteins were transferred onto nitrocellulose in aTransfer box at 22 V run overnight at 4° C. The nitrocellulose wasblocked using BLOTTO A(R) for 45 minutes at room temperature, reactedfirst with primary antibody for 45 minutes at room temperature, followedby washing and reaction with the appropriate secondary antibodyconjugated to horseradish peroxidase. After washing Amersham ECLreagents were used for detection.

The results of a first screen of B cell hybridomas generated from miceimmunized as described above are shown in FIG. 1. The method of thescreen was cellular ELISA testing the binding of the supernatantsremoved from B cell hybridomas cultures to a pool of human plasmacytomacells in one well as compared with their binding to human myelogenicleukemia cell line (K562, serving as a control). Net binding wascalculated as the absorbance recorded for binding to the pool of humanplasmacytoma cells after subtraction of binding to K562 control cellline. In the initial screen a pool of 4 hybridomas was tested in eachwell. Hybridoma pools that recorded high level of differential bindingwere selected and tested individual testing.

No binding was detected to cells from a fresh breast cancer tumor, aprostate cancer cell line, a neuroblastoma cell line, nor to a cervicalcancer cell line. No binding was found to an EBV-transformed B celltumor.

FIG. 2 presents the result of selected hybridomas for the second screen.Numbers 69, 75, and 194 showed specificity as compared to K562 cells.

In a further analysis of the above data, the net binding values (O.D.)obtained for the first screen in comparison with the second screen areshown in FIG. 3.

EXAMPLE 2 Specificity Assessment of Monoclonal Antibodies

Cell surface staining using a panel of monoclonal antibodies Jul. 16,1999 analyzed by Flow cytometry is depicted in FIG. 4. A strong stainingof plasmacytoma cells by MA69 is demonstrated in panel F, while negativestaining was demonstrated for control cell lines, including human B celltumor lines IM9 and HT (IM9 with isotype control, panel A, IM9 withMA69, panel B; HT with isotype control, panel C; HT with MA69, panel D)and myeloma cell line U266 with an isotype control monoclonal antibody(panel E). Furthermore, peripheral blood cells (PBMC) from normalindividuals showed insignificant binding to the antibodies.

Hybridoma cell line IMM002.69.47.4 which produces monoclonal antibodyMA69 was deposited on Aug. 3, 1999, with the American Type CultureCollection, 10801 University Boulevard, Manassas Va. 20110-2209, and hasbeen assigned PTA-450.

EXAMPLE 3 Binding of Monoclonal Antibody to Cell Surface Glycoprotein

B cell hybridoma culture designated MA69 was shown to detect a distinctband on myeloma membranes using Western blots from five human multiplemyeloma cell lines (RPMI1860, U266, KR-12, OPM-1 and NCI H929). Four offive myeloma cell lines showed binding to a cell surface glycoproteinwith an estimated molecular weight of between about 78 and about 120kDa. The PBMC serving as a negative control did not show binding to theantibody, nor was their staining of 2 human B cell tumors (HT and IM9)(FIG. 5).

FIG. 6 presents graphically the results of a repeat of the experimentdescribed in FIG. 5 using a cellular ELISA method. In this experiment,the MA69 detected a distinct band on 5 out of 5 myeloma cell lines withvarying intensity. The control membrane preparations consisting ofnormal PBMC and a human B cell tumor (HT) were not stained by theantibody.

EXAMPLE 4 Detection of Shed Surface Glycoprotein from Cultured MyelomaCells

Human myeloma cells were grown in AIM V serum-free medium for 5 days.The medium was collected and concentrated ten-fold using a Centricondevice (Amersham). As a control, a cell lysate was prepared from the MMcultured in vitro, and fractionated by SDS-PAGE alongside theconcentrated, growth medium. Blotting and probing with MA69 demonstratedthe presence of the surface glycoprotein in the medium (FIG. 7, leftlane).

EXAMPLE 5 Surface Glycoprotein Present on the Surface of Human OvarianCancer Cells

Three ovarian cancer tumors from 3 patients were digested with trypsinand homogenized. The cell lysates were fractionated by SDS-PAGE. The gelwas blotted and probed with MA69. As shown in FIG. 8, the surfaceglycoprotein of the invention is expressed on these cells. The antigenon ovarian cancer cells is a single, glycosylated polypeptide with amolecular weight of about 76 kDa to about 213 kDa as determined by SDSPAGE under reducing conditions. It shares at least one epitope with theaforementioned multiple myeloma surface antigen.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A monoclonal antibody, deposited under ATCCdesignation number PTA-450, or antigen binding fragment thereof, whereinsaid monoclonal antibody is immunoreactive with cell surface membranesof both human myeloma cells and ovarian cancer cells, and is notimmunoreactive with cell surface membranes of human peripheral bloodmononuclear cells, human B cells, neuroblastoma cells, human B cellmyelogenic leukemia cells, breast cancer cells, prostate cancer cells,or cervical cancer cells.
 2. The monoclonal antibody, or bindingfragment thereof, according to claim 1, wherein said antigen bindingfragment is selected from the group consisting of F(ab′)₂, Fab′, Fv, Fd′and Fd.
 3. Hybridoma cell line which is deposited under ATCC AccessionNo. PTA-450.
 4. The monoclonal antibody, or antigen binding fragmentthereof, according to any one of claims 1 or 2 bound to a solid support.5. A composition comprising the monoclonal antibody, or antigen bindingfragment thereof, according to any one of claims 1 or 2 together with aphysiologically acceptable carrier or diluent.